The objectives of this project in this reporting period were i) the cloning and DNA sequencing of the gene (pyrB) encoding aspartate transcarbamylase (ATCase) and the analysis of the gene product, ii) the cloning and DNA sequencing of a C. burnetii insertion sequence (IS), iii) the development of a physical map of the C. burnetii chromosome, iv) the analysis of the induction of heat-shock proteins, and v) the analysis of DNA synthesis during acid-activation of C. burnetii. A. The pyrB gene encodes a catalytically active ATCase that is homologous with other ATCases only at the active site and without a regulatory subunit. B. The IS of C. burnetii is similar though not identical to other bacteria and present in 15-17 copies per genome. C. Variant chromosomal banding among strains correlates with degrees of virulence of C. burnetii. D. Heat-shock (or stress) proteins which are metabolic signals of temperature shifts are synthesized and a 62 kDa protein is exported. E. The condition of acid activation triggers the synthesis of both chromosomal and plasmid DNA. Significance. i) C. burnetii ATCase, as predicted by the DNA sequence of pyrB, is not highly regulated at the level of enzyme activity. Aspects of developmental control of ATCase synthesis can now be studied with the pyrB gene probe. ii) The IS of C. burnetii appears to be unique and with an unusually high number of copies per genome. The encoded basic protein has characteristics of a DNA binding protein which may facilitate genetic recombination experimentation. iii) Analysis of the physical maps of C. burnetii revealed a genome size of 1750 Kb pairs and differences among various strains of known virulence. iv) C. burnetii responds to an increase in temperature and acid activation by synthesizing a variety of heat-shock proteins, chromosomal and plasmid DNA. These markers of metabolic activation will be used to study the adaptation of C. burnetii to life in the phagolysosome.